Summary of Work: The aim of these studies is to determine the mechanisms that regulate developmental expression of genes in male germ cells and the roles of the proteins they encode in gamete development and function. The focus is on genes encoding proteins that are either unique to spermatogenic cells, are germ cell-specific members of a protein family, or produce spermatogenic cell-specific alternative transcripts and protein isoforms. These studies use the gene knockout approach and yeast two-hybrid assays to examine the roles of some proteins believed to be important in male gamete development and function. One example is fertilin beta, a sperm surface glycoprotein which is a member of the ADAM (a disintegrin and metalloprotease domain) family. It has been found that fertilin beta has a key role in sperm-egg interaction and binds to an alpha 6-beta 1 integrin on the egg surface. Mice heterozygous for a targeted mutation in the fertilin beta gene are deficient in sperm-egg membrane adhesion, sperm-egg fusion, migration from the uterus into the oviduct, and binding to the egg zona pellucida. Other examples are protamines 1 and 2, highly basic nuclear proteins that replace the histones and are thought to be essential for DNA compaction in the absence of nucleosomes during sperm development. They have been shown to have developmentally and transcriptionally regulated expression in spermatids. Chimeric mice have been produced that carry a targeted mutation in the protamine 1 or 2 gene and are being mated to produce heterozygous offspring. It is expected that disruption of one or both genes will lead to abnormal nuclear compaction and infertility in homozygous males. A final example is glyceraldehyde 3-phosphate dehydrogenase, a key glycolytic enzyme. A germ cell homolog (Gapd-s) is expressed only in spermatids and has a key role in regulating the generation of ATP required for fertilization. The enzyme is also the likely target for (S)-3-chlorolactaldehyde, a male reproductive toxicant that is a metabolite of the industrial solvent epichlorohydrin. Studies with transgenic mice determined that sequences required for correct expression of Gapd-s are within 230 bp of the transcription initiation site. A knock-out construct for Gapd-s is being prepared and the predicted phenotype of the knock-out is normal appearing sperm that are unable to activate glycolysis, achieve hyper-activated motility, or to fertilize eggs.